Imperial College/14 August 2008

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14 August 2008

Wet Lab

Cloning

  • Parts taken from the registry and transformed by electroporation into XL1-Blue E.coli along with XL1-Blue controls and grown on Kanamycin and Ampicillin plates. Parts taken:
    • C0012 (LacI)
    • J31005 (Chloraphemicol acetyltransferase)
    • B0015 (Double terminator)
    • J04630 (GFP and Double Terminator)
    • I13401 (mRFP and Double Terminator)

B.subtilis

  • The transformation protocol 2 was carried out today, Click link here for protocol
  • The basic principle of this protocol is that competent cells are prepared by growing a culture to a high O.D.600 and then performing electroporation on these competent cells.
  • Today we grew the competent cells, we used an O.D.600 of 1.5 before we harvested them. In addition electroporation was carried out using the plasmid pDR110. Previously we had mini-preped this to give a stock of 40ng/ul. We transformed with 40ng, 120ng, 200ng and 400ng in a volume of 10ul (water was used to make up to 10ul).
  • Transformed cells were plated out and placed into the 30oC incubator.

Dry Lab

  • Completed A More Complex Example of Bayesian Parameter Estimation of Tutorial 2.
  • Sourced for better tracking algorithm, found SpotTracker which is more accurate than ParticleTracker.
  • Went for microscope training, obtained 2 x videos on B.Subtilis motility, stored on server with James.
  • Discussion with Dr. Suhail ( from Structural Bioinformatics Group,Imperial College London) on the computing requirements to analyse the cells motility. We will be allocated a linux 64-bit workstation, that we will be able to access remotely via SSH.

Microscope

  • Chris, Clinton, James and Prudence received microscope training on Widefield 1, recording two 60s videos of B.subtilis swimming for analysis
  • Determined that best results would be obtained by bringing a B.subtilis overnight culture to the microscope facility and diluting 100 fold.